1) Field of the Invention
This invention relates to a method for the quantitative determination of the specific activity of proteinase inhibitors. In one aspect, the invention is directed to the quantitative determination of inactive α1 Proteinase Inhibitor in bodily fluids such as human serum or plasma.
2) Background Art
Emerging evidence suggests a functional link between proteinases and cell signaling. As would be predicted, activation and inactivation of proteinase-activated receptors are selective, e.g., the thrombin-activated receptor can be inactivated by cathepsin G or neutrophil elastase. Examination of the influence of proteinase inhibitors on proteinase-activated receptors is complicated by the variance in affinities, concentrations, and species of proteinase inhibitors represented in serum. The proteinase inhibitor in serum exhibiting the greatest concentration is α1 proteinase inhibitor (α1PI, α1-antitrypsin), and the proteinase inhibitor encompassing the broadest spectrum is α2 macroglobulin (α2M).
In the acute phase of inflammation, quantitative levels of α1PI have been reported to significantly increase, as do proteolytic fragmentation and proteinase complexation, both of which can diminish the functional capacity of α1PI. The functional capacity of α1PI during the acute phase has not previously been examined. Further, in some situations, the association of α2M with neutrophil elastase in plasma is competitively favored, and in this case elastase-mediated proteolysis of low-molecular-weight peptides and cytokines can persist. The relative concentrations of elastolytic proteinases, the inhibitor α1PI, and the substrate-restricting α2M form a tightly regulated mechanism for discreet targeting of elastase activity.
It has been previously observed using serially diluted serum that the residual uninhibited enzymatic activity of exogenously added elastase exhibits bimodal regulation. The bimodal behavior of serum was demonstrated to result from the dual activities of α1PI and α2M; however, these investigators did not attempt to derive a numerical value for quantitating the active fraction of α1PI. While elastase is completely inhibited by α1PI, association of elastase with α2M excludes its activity except toward low molecular substrates. When the concentration of α2M exceeds that of elastase, catalytic activity if unaffected by adding α1PI since α2M is not replaced by α1PI in these complexes. When the concentration of elastase exceeds that of α2M in this scenario, elastase is available for complexing with added α1PI resulting in a decrease in catalytic activity. Physiologic concentrations of the common phenotypes of α1PI have been approximated as 20-53 μM and that of α2M as 1.56-4.96 μM so that as serum is diluted and incubated with a constant concentration of elastase, the contribution from α2M in elastase protection becomes negligible by this method of detection, and the contribution from α1PI is detected as increased inhibition. On the other hand, as serum becomes excessively dilute, the contribution from α1 PI also becomes negligible to detection resulting in decreased inhibition. Therefore, a serum dilution exists at which minimum catalytic activity can be measured by exploiting the properties of unequal serum concentration and unequal outcomes of complexes between elastase and α1PI and α2M. The maximum reduction in catalytic activity is a measure of the functionally active concentration of α1PI in competition with α2M for elastolytic enzymes. The relationship between reduction in catalytic activity and the precise quantitation of functional α1PI in competition with α2M has not previously been examined.
Proteinase inhibition is only one of the diverse biologic activities of α1PI and α2M including alteration of the cellular effects of polymorphonuclear neutrophils, found that α1PI It decreases antigen-driven, PHA and, Con A, but not PWM, lymphocyte responsiveness. In fact, inhibition of DNA synthesis and proliferation by α1PI has been demonstrated in erythroid progenitor cells and lymphocytes. It has been reported that α1PI deficient serum mediates enhancement of lymphocyte response to PHA and increases zymosan activation of monouclear cells and PMN. The ability to measure the functional capacity of proteinase inhibitors in serum is paramount to determining the interrelationship between proteinase inhibitors and immune responsiveness in pathology. Association rates previously derived using isolated proteinases and inhibitors suggested the feasibility for measuring these activities in serum.
Quantitative determination of serum α1PI has traditionally been performed nephelometrically; however, antigenically quantitated levels may not be representative of functional capacity. It has previously been observed that α1PI in serum exhibits bimodal behavior as the result of various concentrations of proteinase inhibitors, specifically α2macro-globulin (α2M) and inter-a-trypsin inhibitor, which compete in binding to a panel of serine proteinases. Consequently, it has not previously been possible to assign a numerical value for the specific activity of these competing proteinase inhibitors in serum.
In J. Clin. Chem. Clin. Biochem, Vol. 25, 1987, pp. 167-172, M. C. Gaillard, et al disclosed the determination of functional activity of α1 proteinase inhibitors and α2 macroglobulin in human plasma using elastase. They were able to devise an assay method to determine the amounts of functional activity of α1 proteinase inhibitors and α2 macroglobulin respectively in human plasma.
The method of Gaillard, et al employed mixing elastase with α1, proteinase inhibitor and α2 macroglobulin, all three with some degree of purity, but with undetermined activity. Their method gives a result, the equivalence point (Ve) used in calculating “total elastase inhibiting capacity which is defined as the number of ml of plasma required to bind 1 μmol of porcine pancreatic elastase.” While this is an important finding, however, the value for total elastase binding activity is not reproducible using varying sources or concentrations of elastase as demonstrated in Bristow et 1998, Clin. Immunol. Immunopathol. Nor does the method of Gaillard, et al give any information about whether α1 proteinase inhibitor is being degraded or is at steady state. Degradation occurs during inflammation, and it is desirable to detect degradation of α1 proteinase as a prognostic indication of disease state. For this reason, the present invention sought to determine the relationship between the equivalence point described by Gaillard, et al and the number of total molecules, active molecules, and inactive molecules of α1 proteinase inhibitor in serum and other complex body fluids. The experimental method to define this relationship was to observe the change in residual catalytic activity of elastase when a quantifiable number of functioning molecules of elastase were incubated with a quantifiable number of functioning molecules Of α2 macroglobulin before or at the same time as addition of a quantifiable number of functioning molecules of α1 proteinase inhibitor. By varying the three numbers of molecules, it was possible to derive the theoretical relationship between elastase inhibiting capacity and the ratio of elastase bond to α1 proteinase inhibitor or α2 macroglobulin. Detivation of the theoretical relationship between elastase bound to α1 proteinase inhibitor or α2 macroglobulin in competition (herein referred to as the first unique derivation) is unique to the instant method and has not been attempted previously.
However, derivation of a theoretical relationship does not necessarily imply a physiological relationship. Therefore, the theoretical relationship was tested using serum from healthy individuals with known steady state levels of antigenically determined α1 proteinase inhibitor. Application of the first unique derivation to the quantification of serum elastase inhibiting activity allowed the second unique step of the instant method (herein referred to as the second unique derivation), the relationship between the “equivalence point” (Ve in units of plasma volume), the number of elastase molecules added to serum, and residual catalytic activity (in units of elastase molecules).
However, derivation of the relationship between elastase molecules added to serum and residual catalytic activity still does not yield the number of molecules of α1 proteinase inhibitor or α2 macroglobulin in serum. Application of the second unique derivation to sera from a sufficiently large population of individuals allowed the third unique step of the instant method (herein referred to as the third unique derivation), the relationship between residual catalytic activity and the number of functionally active molecules of α1 proteinase inhibitor or α2 macroglobulin.
Of considerable significance, these three unique derivations allow for the first time, detection of degraded or inactive α1 proteinase inhibitor as a prognostic indication of disease state. Prior to the present invention, there has never existed art to measure inactive proteinase inhibitors in complex body fluids.
During inflammation, the total concentration of serum α1 PI proteinase inhibitor increases two-to-four fold. However, the “total elastase inhibiting activity” as determined by the method of Gaillard, et al may or may not remain at steady state. Using the instant method, recently published evidence (Bristow, et al, 2001. Clin. Diagn. Lab. Immnunol. 8: p. 938) discloses that inactive α1 proteinase inhibitor is strongly correlated with HIV disease progression (p<3×10−8). Importantly, in AIDS, the total and active concentrations of α1 proteinase inhibitor were not statistically different from normal, but inactive α1 proteinase inhibitor was significantly elevated (p<0.0001). These data are compelling that detecting inactive al proteinase inhibitor using the instant method provides a prognostic indicator in disease progression for which there is no prior art.
By applying known constants representing the association of proteinase inhibitors with porcine pancreatic elastase (PPE), the theoretical relationship between the functional and antigenic values for α1 PI and α2 M has been empirically derived allowing, for the first time, the calculation of their specific activities in serum. The serum concentration of α1PI was found to be highly correlated with residual uninhibited PPE catalytic activity in healthy individuals, but not in individuals exhibiting fragmented or complexed α1PI. Using these techniques, both the antigenic and functional levels of α1PI were determined in sera from subjects with insulin-dependent diabetes mellitus (IDDM) who has been clinically diagnosed as having either periodontal disease or gingival health. Determination of quantitative levels by antigen-capture suggests that the IDDM subjects with periodontitis manifest dramatically increased levels of fragmented serum α1PI compared with their orally healthy counterparts or normal controls.
The following abbreviations are employed in the specifications and amended claims:                PPE—porcine pancreatic elastase        α1PI—α1 proteinase inhibitor (α1-antitrypsin)        α2M—α2 Macroglobulin        HNE—human neutrophil elastase        IαI—inter-α-trypsin inhibitor        APE—porcine pancreatic elastase        PBS—0.01M phosphate, 0.15M NaCl, pH 7.2        TBS—0.05 M Tris, 0.15M NaCl, pH 7.8        SaqNA—succinyl-L-Ala-L-Ala-L Ala p-Nitro-anilide.        EDTA—ethylenediaminotetraacetic acid        ACD—acetate-citrate-dextrose        IDDM—insulin-dependent diabetes anellites        